CN112063605B - Method for preparing gentiooligosaccharide by catalyzing cellulose with complex enzyme and application of method - Google Patents

Method for preparing gentiooligosaccharide by catalyzing cellulose with complex enzyme and application of method Download PDF

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CN112063605B
CN112063605B CN202011015802.1A CN202011015802A CN112063605B CN 112063605 B CN112063605 B CN 112063605B CN 202011015802 A CN202011015802 A CN 202011015802A CN 112063605 B CN112063605 B CN 112063605B
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吴敬
夏伟
许俊勇
徐星豪
黄燕
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Jiangnan University
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    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source

Abstract

The invention discloses a method for preparing gentiooligosaccharide by catalyzing cellulose with complex enzyme and application thereof, belonging to the technical field of enzyme engineering. The invention obtains the mutant by a site-directed mutagenesis mode on the basis of beta-glucosidase from Thermotoga sp.KOL6, and realizes the high-efficiency expression of the mutant gene in pichia pastoris by taking pichia pastoris KM71 as an expression host. Meanwhile, the complex enzyme can effectively convert cellulose into gentiooligosaccharide; when the gentiooligosaccharide is prepared by taking phosphoric acid swelling cellulose and glucose as substrates and performing enzyme catalytic conversion, and the cellulase and the beta-glucosidase mutant Q34G/Y200F are compounded, the total conversion rate of the raw materials is 19.1 percent, and is respectively improved by 74.7 percent compared with the wild beta-glucosidase. The technical scheme of the invention has high industrial application value.

Description

Method for preparing gentiooligosaccharide by catalyzing cellulose with complex enzyme and application of method
Technical Field
The invention relates to a method for preparing gentiooligosaccharide by catalyzing cellulose with complex enzyme and application thereof, belonging to the technical field of enzyme engineering.
Background
The gentiooligosaccharide is an oligosaccharide formed by connecting glucose with beta-1, 6 glycosidic bonds, the main component is gentiobiose, and the gentiotriose and gentiotetraose contain a small amount, so that the gentiooligosaccharide is a novel functional oligosaccharide. The gentiooligosaccharide has excellent intestinal tract benefiting effect, has the characteristics of low viscosity, good water binding capacity, very stable pH and heat and the like compared with functional oligosaccharides such as isomaltooligosaccharide, fructo-oligosaccharide, galacto-oligosaccharide and the like, and can be applied to foods which are not tolerant by other oligosaccharides; and has unique refreshing bitter taste, and can increase the richness of food taste. The low-polymer gentiooligosaccharide has wide application in popular foods at present and is a high-added-value saccharide product with great development prospect.
So far, only Japan food chemical company produces the product (trade names Gentose #45 and Gentose #80) at home and abroad, and the yield is not high, only 350 tons, and the market gap is large. With the expansion of market demand, the preparation of the gentiooligosaccharide by the enzyme method has the advantages of mild reaction conditions, safe strains, small pollution, low cost, easy separation and the like, is concerned by researchers at home and abroad, is the main development trend of the prior production of the gentiooligosaccharide, and has no more ideal case report at present. Early researchers utilized beta-glucosidase to catalyze reverse hydrolysis condensation reaction, and glucose syrup obtained by hydrolyzing starch with high concentration glucose or acid is taken as a substrate to prepare the gentiooligosaccharide, and the conversion rate is approximately distributed between 5% and 15% under the substrate concentration of 200-900g/L glucose. In recent years, researches on producing gentiooligosaccharide by using beta-glucosidase transglycosylation reaction and taking cellobiose as a substrate gradually appear, and compared with the current reaction system taking high-concentration glucose as a substrate, the transglycosylation reaction system has obvious advantages in catalytic efficiency and conversion rate, the conversion rate is about 19.6%, but the substrate cellobiose is expensive in cost and is not suitable for large-scale production; how to develop a method which can effectively utilize a cheap substrate and can efficiently prepare the gentiooligosaccharide is an urgent problem to be solved.
Disclosure of Invention
The invention establishes a reaction system for preparing gentiooligosaccharide by converting cellulose under the catalysis of complex enzyme, under the condition that glucose with certain concentration inhibits the hydrolysis activity of beta-glucosidase, the beta-glucosidase with high transglycosidation activity is used for replacing the beta-glucosidase with high hydrolysis activity in the traditional cellulose degradation enzyme system, cellobiose can be converted into gentiooligosaccharide, and the product inhibition of cellobiose is eliminated; the upstream cellulase hydrolyzes cellulose to fill the consumption of cellobiose. The high-efficiency preparation of the gentiooligosaccharide can be realized by regulating and controlling a synergistic reaction system.
The invention firstly provides a beta-glucosidase mutant, which is obtained by mutating the 34 th site and/or the 200 th site of beta-glucosidase with an amino acid sequence shown as SEQ ID NO. 1.
In one embodiment of the present invention, the β -glucosidase mutant is: the amino acid sequence is shown as SEQ ID NO.1, the 34 th site of the beta-glucosidase is obtained by mutating glutamine into glycine, and the name is Q34G;
or the 34 th site of the beta-glucosidase with the amino acid sequence shown as SEQ ID NO.1 is obtained by mutating glutamine into methionine and is named as Q34M;
or the 200 th site of the beta-glucosidase with the amino acid sequence shown as SEQ ID NO.1 is obtained by mutating tyrosine into phenylalanine and is named as Y200F;
or the 34 th site of the beta-glucosidase with the amino acid sequence shown as SEQ ID NO.1 is mutated into glycine from glutamine, and the 200 th site of the beta-glucosidase with the amino acid sequence shown as SEQ ID NO.1 is mutated into phenylalanine from tyrosine, and the beta-glucosidase is named as Q34G/Y200F.
In one embodiment of the invention, the amino acid sequence of the β -glucosidase mutant Q34G is shown as SEQ ID No.9, the amino acid sequence of the β -glucosidase mutant Q34M is shown as SEQ ID No.10, the amino acid sequence of the β -glucosidase mutant Y200F is shown as SEQ ID No.11, and the amino acid sequence of the β -glucosidase mutant Q34G/Y200F is shown as SEQ ID No. 12.
In one embodiment of the invention, the nucleotide sequence encoding the beta-glucosidase is shown as SEQ ID NO. 2.
The invention also provides a gene for coding the beta-glucosidase mutant.
The invention also provides an expression vector carrying the gene.
The invention also provides a microbial cell carrying the gene or the expression vector.
In one embodiment of the invention, the microbial cell is a bacterium or a fungus.
The invention also provides a method for preparing gentiooligosaccharide by multienzyme compounding, which comprises the steps of adding cellulase and the beta-glucosidase mutant into a reaction system containing cellulose and glucose for reaction to obtain a reaction solution; and separating the reaction solution to obtain the gentiooligosaccharide.
In one embodiment of the invention, the cellulase is one or more of an endocellulase, a β -glucanase, a cellobiohydrolase type I, a cellobiohydrolase type II.
In one embodiment of the present invention, the cellulase is added to the reaction system at a concentration of 10 to 50U/g cellulose filter paper enzyme activity.
In one embodiment of the present invention, the cellulase is added to the reaction system at a concentration of 25 to 50U/g cellulose filter paper enzyme activity.
In one embodiment of the present invention, the concentration of the beta-glucosidase mutant added to the reaction system is 400U/g glucose 100-.
In one embodiment of the present invention, the concentration of the beta-glucosidase mutant added to the reaction system is 300-400U/g glucose.
In one embodiment of the present invention, the concentration of the substrate cellulose in the reaction system is 200 g/L.
In one embodiment of the present invention, the concentration of glucose as a substrate in the reaction system is 100g/L to 400 g/L.
In one embodiment of the invention, the temperature of the reaction is 60 ℃.
In one embodiment of the invention, the pH of the reaction is 5.0.
In one embodiment of the invention, the reaction time is 24 to 96 hours.
In one embodiment of the invention, the reaction time is 96 h.
In one embodiment of the present invention, the substrate cellulose is treated by: the phosphoric acid swelling cellulose is obtained by carrying out phosphoric acid swelling pretreatment on the cellulose raw material.
In one embodiment of the present invention, the method is: adding cellulose swelled by phosphoric acid into buffer solution with the pH value of 5.0 to the concentration of 200g/L, adding glucose to the concentration of 400g/L to obtain a reaction system, adding 50U/g cellulose filter paper enzyme activity cellulase and the 400U/g glucose beta-glucosidase mutant into the reaction system, and reacting for 96 hours at the temperature of 60 ℃.
In one embodiment of the present invention, the buffer is a citrate-phosphate buffer.
The invention also provides the application of the mutant or the gene or the expression vector or the microbial cell or the preparation method in preparing the gentiooligosaccharide.
Advantageous effects
(1) The complex enzyme can effectively convert cellulose into gentiooligosaccharide; when 20% Phosphoric Acid Swelling Cellulose (PASC) and 40% glucose are used as substrates for enzyme catalytic conversion to prepare gentiooligosaccharide, cellulase and beta-glucosidase mutant Q34G, Q34M, Y200F or Q34G/Y200F are compounded, the total conversion rate is 17.6%, 16.0%, 16.7% and 19.1% respectively, and is improved by 60.6%, 46.1%, 52.7% and 74.7% respectively compared with wild beta-glucosidase. The compound enzyme (cellulase + beta-glucosidase mutant Q34G/Y200F) with the highest conversion rate takes cellulose and glucose as raw materials to prepare the gentiooligosaccharide with the highest yield of 114.7g/L and the cellobiose conversion rate of 57.3 percent.
(2) The invention realizes the preparation of the gentiooligosaccharide by using a more extensive and cheap cellulose substrate, and greatly reduces the production cost of the gentiooligosaccharide in industry, so the technical scheme of the invention has high industrial application value.
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FIG. 1: schematic diagram of preparation of gentiooligosaccharide by catalyzing cellulose with compound enzyme.
Detailed Description
Cellulose, glucose, is referred to in the following examples as available from Shanghai Vitta Chemicals, Inc.; escherichia coli JM109 competent cells were purchased from Shanghai, and Pichia pastoris KM71 was purchased from Invitrogen; trichoderma reesei was purchased from China culture Collection.
The media involved in the following examples are as follows:
LB liquid medium: 5g/L of yeast powder, 10g/L of tryptone and 10g/L of NaCl.
MD solid medium: YNB 13.4g/L, biotin 4X 10-4g/L, glucose 20g/L and agar 20 g/L.
YPD liquid medium: peptone 20g/L, yeast extract 10g/L, glucose 20 g/L.
YPD solid Medium: 20g/L agar was added to the YPD liquid medium.
BMGY medium: YNB 13.4g/L, glycerol 10g/L, biotin 4X 10-4g/L, 0.1mol/L potassium phosphate buffer solution (pH 6.0), peptone 20g/L, yeast powder 10 g/L.
BMMY medium: YNB 13.4g/L, methanol 1%, biotin 4X 10-4g/L, 0.1mol/L potassium phosphate buffer solution (pH 6.0), peptone 20g/L, yeast powder 10 g/L.
Fermenting a seed culture medium: 5.0g/L yeast powder, 10.0g/L tryptone, 10.0g/L glucose and 30g/L glycerol.
BSM medium: 85% phosphoric acid 26.7mL/L, CaSO4 0.93g/L，K2SO4 18.2g/L，MgSO4·7H214.9g/L of O, 4.13g/L of KOH, 30.0g/L of glycerol and 4.32mL/L of trace element salt solution.
The detection methods referred to in the following examples are as follows:
and (3) enzyme activity determination of cellulase filter paper:
punching a round filter paper chip with the radius of 0.5cm on Waterman filter paper by a puncher, putting 10 round filter paper chips into 900 mu L of buffer solution with proper pH, then putting the filter paper chips into a water bath kettle at a certain temperature for preheating for 5min, then adding 100 mu L of enzyme solution with certain dilution times, accurately timing for 3h, adding 1500 mu L of DNS to terminate the reaction, measuring the light absorption value at 540nm after bathing for 5min in a boiling water bath, and measuring the blank control group by using the enzyme solution inactivated by heat treatment in the same reaction system.
The enzyme activity unit is defined as: the enzyme activity to hydrolyze a cellulosic substrate to produce 1. mu. mol of reducing sugars per minute is one filter paper enzyme activity unit (FPU).
And (3) measuring the enzyme activity of the beta-glucosidase:
the reaction system was 1mL of acetic acid buffer (960. mu.L, pH 5.0) to which was added crude enzyme solution diluted moderately20 mu L, adding 100mmol/L pNPG 20 mu L, reacting in 60 deg.C constant temperature water bath for 10min, immediately adding 1mol/L Na 200 mu L after 10min2CO3The reaction was stopped with ice bath for 5min and the absorbance was measured at 405 nm. The enzyme solution inactivated by heating was treated as a blank in the same manner.
The beta-glucosidase enzyme activity is defined as: the enzyme activity of 1 mu mol of p-nitrophenol generated by hydrolyzing pNPG per minute per milliliter of enzyme solution is one enzyme activity unit.
And (3) detecting the content of the gentiooligosaccharide:
detecting the content of gentiooligosaccharide by HPLC;
the chromatographic conditions were as follows: an Agilent 1200HPLC chromatograph, an Agilent autosampler, and a RID differential detector; the mobile phase is 75% acetonitrile, and the flow rate is 0.8 mL/min; the column temperature was 35 ℃.
Example 1: construction of beta-glucosidase mutant
According to the gene sequence of beta-glucosidase with the nucleotide sequence shown as SEQ ID NO.2, primers for introducing Q34G, Q34M and Y200F mutation are designed and synthesized, the beta-glucosidase gene is subjected to site-specific mutagenesis, a DNA coding sequence is determined, and the 34 th Gln codon is changed into Gly codon, the 34 th Gln codon is changed into Met codon, and the 200 th Tyr codon is changed into Phe codon. And (3) placing the mutant gene in an expression vector and introducing Pichia pastoris for expression to obtain the single-mutation beta-glucosidase. Single mutation Q34G, Q34M, Y200F and combined mutation Q34G/Y200F are constructed by using an expression vector pPIC9K-TpBgl3A (described in the Chinese patent application text with the publication number of CN 111500560A) as a template by using a rapid PCR (polymerase chain reaction) technology.
Site-directed mutagenesis primers for introducing the Q34G mutation were:
a forward primer: 5' -ACGGGAACAAGTTGGGGGAACGGTTCTTGTGTA-3’(SEQ ID NO.3)
Reverse primer: 5' -TACACAAGAACCGTTCCCCCAACTTGTTCCCGT-3’(SEQ ID NO.4)
Site-directed mutagenesis primers for introducing the Q34M mutation were:
a forward primer: 5' -CAAGTTGGATGAACGGTTCTTGTGTAGGCAACA-3’(SEQ ID NO.5)
Reverse primer: 5' -GAACCGTTCATCCAACTTGTTCCCGTCACGATG-3’(SEQ ID NO.6)
The site-directed mutagenesis primers for introducing the Y200F mutation were:
a forward primer: 5' -TGTGTGCTCGTGGAATAAAGTGAATGGTACTTATA-3’(SEQ ID NO.7)
Reverse primer: 5' -ACTTTATTCCACGAGCACATCACGCTGGCGACG-3’(SEQ ID NO.8)
The PCR reaction systems are as follows: 5 XPS buffer 10. mu.L, dNTPs Mix (2.5mM) 4. mu.L, forward primer (10. mu.M) 1. mu.L, reverse primer (10. mu.M) 1. mu.L, template DNA 1. mu.L, Primerstar HS (5U/. mu.L) 0.5. mu.L, and double distilled water was added to 50. mu.L.
The PCR amplification conditions were: pre-denaturation at 94 ℃ for 4 min; followed by 30 cycles (98 ℃ for 10s, 55 ℃ for 15s, 72 ℃ for 10 min); extension was continued for 10min at 72 ℃.
Digesting the PCR product by Dpn I, transforming escherichia coli JM109 competent cells to obtain a transformation product, culturing the transformation product in an LB culture medium (containing 100 mug/mL ampicillin) overnight, selecting a positive clone, culturing the positive clone in an LB liquid culture medium (containing 100 mug/mL ampicillin), extracting plasmids, correctly sequencing all mutant plasmids, transforming and expressing host pichia pastoris KM71 competent cells by the mutant plasmids, selecting 96 single colonies on the MD solid culture medium, dibbling the colonies on a new MD solid culture medium at equal intervals, culturing the colonies for 1-2 days at 30 ℃, transferring all the colonies to a deep-hole plate filled with 1mL of BMGY culture medium to culture the strains for 48 hours, centrifuging, abandoning the supernatant, suspending the strains by using 0.5mL of BMMY culture medium, adding 1% (v/v) of methanol to induce the strains for 2 days, centrifuging the strains to obtain the supernatant for enzyme activity, selecting a transformant with the highest enzyme activity and storing the transformant at the temperature of minus 80 ℃; the obtained recombinant bacteria are respectively named as KM71/pPIC9K-Q34G, KM71/pPIC9K-Q34M, KM71/pPIC9K-Y200F and KM71/pPIC 9K-Q34G/Y200F.
Example 2: expression of wild type and mutant beta-glucosidase and determination of specific enzyme activity
A recombinant Pichia pastoris strain KM71/pPIC9K-TpBgl3A (described in the Chinese patent application publication No. CN 111500560A) capable of expressing wild-type beta-glucosidase.
Taking the recombinant strain KM71/pPIC9K-TpBgl3A and the recombinant strain KM71/pPIC9K-Q34G, KM71/pPIC9K-Q34M, KM71/pPIC9K-Y200F and KM71/pPIC9K-Q34G/Y200F obtained in example 1 out of a refrigerator, respectively taking 30 mu L of bacterial liquid from the glycerol tube, inoculating the bacterial liquid into 10mL of YPD liquid culture medium, carrying out shake culture in a shaker at 30 ℃ for 24-36 h to obtain culture liquid, respectively taking 2.5mL of the culture liquid, transferring the culture liquid into 50mL of BMGY culture medium, carrying out 24h culture at 30 ℃, centrifuging at 4000rpm for 10min, filtering and collecting thalli, suspending the thalli with 25mL of BMMY culture medium containing 1% (v/v) methanol, once every 24h, wherein the final concentration of the methanol is 1% (v/v), and carrying out culture at 120 ℃ to obtain fermentation liquid; centrifuging the obtained fermentation liquid at 10000rpm for 15min, and taking the supernatant to obtain a crude enzyme liquid containing wild enzyme and mutants Q34G, Q34M, Y200F and Q34G/Y200F.
The specific activities of the catalytic hydrolysis reactions of the crude enzyme solutions are respectively measured, and the specific activities of the hydrolysis reactions of the obtained beta-glucosidase single mutant enzymes are listed in table 1, wherein the specific activities of the hydrolysis reactions of the mutant Q34M are slightly increased, the specific activities of the hydrolysis reactions of the mutant Q34G are basically equal to those of the wild type, and the specific activities of the hydrolysis reactions of the mutant Y200F and the combined mutant Q34G/Y200F are respectively reduced to 2.6% and 2.1% of the wild type. The reduction of the enzymatic hydrolysis activity is beneficial to the occurrence of the transglycosidation reaction and the accumulation of the transglycosidation product.
TABLE 1 enzyme Activity of different beta-glucosidases in crude enzyme solutions
Figure BDA0002699016910000061
Example 3: method for preparing gentiooligosaccharide by catalyzing cellulose with compound enzyme
The method comprises the following specific steps:
pretreated cellulose and glucose are used as raw materials, and the gentiooligosaccharide is prepared by multi-enzyme composite catalytic conversion (as shown in figure 1).
1. The cellulose pretreatment conditions were as follows: the preparation method of Phosphoric Acid Swelling Cellulose (PASC) obtained by expanding and pretreating a cellulose raw material with Phosphoric acid refers to Schiilein et al (Reference: Schulein. M. enzymic properties of cellulose from Humicola insolens [ J ]. Biotechnol,1997,57(1-3):71-81) with proper adjustment. The method comprises the following specific steps:
(1) weighing 10g of microcrystalline cellulose Avicel, adding 30mL of deionized water, and fully stirring; slowly adding 500mL of precooled 86.2% phosphoric acid solution while stirring; fully stirring under the ice bath condition until the solution is transparent and viscous and has no particles;
(2) transferring the solution obtained in the step (1) to a temperature of 4 ℃ and standing for 12 hours to fully expand the cellulose;
(3) adding 1L of ice water into the puffed cellulose obtained in the step (2), and stirring while adding to obtain a white floccule to obtain a mixture;
(4) centrifuging the mixture obtained in the step (3) at 6000rpm for 20min, collecting white flocculent precipitate, and discarding the supernatant (big centrifuge cup);
(5) repeating the steps (3) and (4); to the resulting white flocculent precipitate was added 10mL of 2M Na2CO3Fully stirring and neutralizing the residual phosphoric acid;
(6) adding 1L of ice water into the step (5), fully stirring, centrifuging at 6000rpm for 20min, and removing the supernatant;
(7) and (5) repeating the step (6) until the pH value of the solution reaches 5-7, drying the precipitate, namely the phosphoric acid swelling cellulose, and storing the prepared phosphoric acid swelling cellulose PASC at 4 ℃ for later use.
2. Preparation of cellulase
The cellulase is obtained by fermenting trichoderma reesei, contains endo-cellulase and cellobiohydrolase activity, and is prepared by the following specific steps:
(1) washing Trichoderma reesei spore stored on PDA slant with sterile water to obtain spore with concentration of 107cfu/mL spore suspension; wherein, PDA culture medium: 200g/L of potato, 20g/L of glucose and 20g/L of agar;
(2) inoculating the spore suspension into a 1L shake flask filled with 200mL of fermentation medium according to the inoculation amount of 2% (v/v), stirring at the rotation speed of 200r/min, pH of 5.0, and culturing at 30 ℃ for 6d to obtain a fermentation liquid; whereinAnd fermentation medium: 18g/L lactose, 10g/L microcrystalline cellulose, 12g/L corn starch, (NH4)2SO4 0.5g/L，MgSO4 1g/L，CaCl20.5g/L, 1mL/L of Mandels trace element nutrient salt, 802 mL/L of Tween and pH 4.8.
(3) And (3) centrifuging the fermentation liquor obtained in the step (2) to obtain supernatant, namely crude cellulase liquid, and measuring the filter paper enzyme activity of the crude cellulase liquid, wherein the enzyme activity is 456 FPU/mL.
3. Method for preparing gentiooligosaccharide by using compound enzyme
Adding the phosphate swelling cellulose (PASC) prepared in the step 1 to a 50mM citric acid-phosphate buffer solution with the pH value of 5.0 to obtain a 200g/L concentration solution, adding glucose to a 400g/L concentration solution, adding the crude cellulase enzyme solution obtained in the step 3 to obtain a 50U/g cellulose filter paper enzyme activity (FPU) concentration solution, and adding the crude beta-glucosidase enzyme solution obtained in the example 2 to obtain a 400U/g glucose concentration solution to obtain a reaction system; the reaction system is placed in a water bath shaker at 60 ℃ and 150rpm for reaction for 96h, samples are taken once every 24h, the samples are centrifuged at 12000rpm for 10min, and the supernatant is diluted appropriately, filtered by a 0.45 mu m ultrafiltration membrane and analyzed by HPLC.
The detection results are shown in table 2, when beta-glucosidase mutant Q34G, beta-glucosidase mutant Q34M, beta-glucosidase mutant Y200F and beta-glucosidase mutant Q34G/Y200F are respectively adopted to prepare the gentiooligosaccharide by catalyzing cellulose and glucose by the compound enzyme, the total conversion rate is respectively 17.6%, 16.0%, 16.7% and 19.1%, and the total conversion rate is respectively improved by 60.6%, 46.1%, 52.7% and 74.7% compared with that of wild type beta-glucosidase.
The complex enzyme (cellulase + beta-glucosidase mutant Q34G/Y200F) with the highest conversion rate has the advantages that the highest yield of the gentiooligosaccharide prepared by taking cellulose and glucose as raw materials can reach 114.7g/L, the conversion rate of cellobiose can reach 57.3%, and the application value is certain.
TABLE 2 yield of gentiooligosaccharide production by wild enzyme and mutant
Figure BDA0002699016910000081
Example 4: effect of the Source of beta-glucosidase on the yield of gentiooligosaccharide
The specific implementation manner is the same as that of example 3, except that the beta-glucosidase is adjusted to: wild enzyme of beta-glucosidase from trichoderma viride (described in ZL201710485406.7 chinese patent application text); the total conversion rate was: 8.7 percent.
Example 5: influence of compounding ratio of enzyme on yield of gentiooligosaccharide
The specific implementation manner is the same as that in example 3, except that the compounding ratio of the cellulase and the beta-glucosidase is adjusted.
When the addition amount of the fixed wild beta-glucosidase is 400U/g glucose, the addition amounts of the cellulase are respectively adjusted to be 10U/g cellulose filter paper enzyme activity (FPU), 15U/g cellulose filter paper enzyme activity (FPU) and 25U/g cellulose filter paper enzyme activity (FPU), the reaction lasts for 96 hours, and the total conversion rate is respectively 5.3%, 5.9% and 8.2%.
When the addition amount of the fixed cellulase is 50U/g cellulose filter paper enzyme activity (FPU), the addition amounts of the wild beta-glucosidase are respectively adjusted to be 100U/g glucose, 200U/g glucose and 300U/g glucose, the reaction is carried out for 96 hours, and the total conversion rate is respectively 3.7%, 4.9% and 9.2%.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> method for preparing gentiooligosaccharide by catalyzing cellulose with complex enzyme and application thereof
<130> BAA200930A
<160> 12
<170> PatentIn version 3.3
<210> 1
<211> 714
<212> PRT
<213> Artificial sequence
<400> 1
Gln Thr Ser Asp Trp Asp Glu Ala Tyr Ser Lys Ala Leu Asp Ser Leu
1 5 10 15
Ala Lys Leu Ser Gln Asn Glu Lys Ile Gly Ile Val Thr Gly Thr Ser
20 25 30
Trp Gln Asn Gly Ser Cys Val Gly Asn Thr Tyr Gln Pro Ser Ser Ile
35 40 45
Asp Tyr Pro Ser Leu Cys Leu Gln Asp Gly Pro Leu Gly Ile Arg Tyr
50 55 60
Ala Asn Pro Val Thr Ala Phe Pro Ala Gly Ile Asn Ala Gly Ala Thr
65 70 75 80
Trp Asp Arg Ser Leu Ile Lys Asp Arg Gly Ala Ala Leu Gly Glu Glu
85 90 95
Ala Lys Ser Leu Gly Val His Val Ser Leu Gly Pro Val Ala Gly Pro
100 105 110
Leu Gly Lys Val Pro Gln Gly Gly Arg Leu Trp Glu Gly Phe Ser Val
115 120 125
Asp Pro Tyr Leu Ser Gly Val Ala Met Thr Glu Thr Ile Asn Gly Val
130 135 140
Gln Gly Ala Gly Ala Gln Ala Cys Ala Lys His Tyr Ile Gly Asn Glu
145 150 155 160
Gln Glu Thr Asn Arg Asn Tyr Ile Asp Ser Thr Ile Asp Asp Arg Ala
165 170 175
Phe His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Asn
180 185 190
Val Ala Ser Val Met Cys Ser Tyr Asn Lys Val Asn Gly Thr Tyr Thr
195 200 205
Cys Glu Asn Pro Ala Val Leu Asn His Thr Leu Lys Thr Glu Leu Gly
210 215 220
Phe Lys Gly Tyr Ile Met Ser Asp Trp Gly Ala Gln His Thr Thr Ser
225 230 235 240
Gly Ser Ala Asn Ala Gly Leu Asp Met Thr Met Pro Gly Ser Asp Leu
245 250 255
Ser Asn Pro Pro Gly Asn Val Leu Trp Gly Gln Lys Leu Ala Asp Ala
260 265 270
Ile Ser Asn Gly Glu Val Glu Gln Ser Arg Leu Asp Asp Met Val Thr
275 280 285
Arg Ile Leu Ala Ala Trp Tyr Leu Val Gly Gln Asp Gln Gly Tyr Pro
290 295 300
Ser Val Gln Phe Asn Ser Trp Asn Gly Gly Gln Thr Ser Ala Asn Val
305 310 315 320
Thr Gly Asp His Ala Thr Val Val Arg Asn Val Ala Arg Asp Ser Ile
325 330 335
Val Leu Leu Lys Asn Asp Asn Asn Thr Leu Pro Leu Ser Lys Pro Asn
340 345 350
Ser Leu Ala Ile Ile Gly Ser Asp Ala Ala Val Asn Pro Asp Gly Pro
355 360 365
Asn Ala Cys Ser Asp Arg Gly Cys Asp Thr Gly Thr Leu Ala Met Gly
370 375 380
Trp Gly Ser Gly Thr Cys Glu Phe Pro Tyr Leu Val Gly Pro Leu Glu
385 390 395 400
Ala Ile Lys Asn Gln Ala Asn Ala Asp Gly Thr Thr Ile Thr Ser Ser
405 410 415
Thr Thr Asp Ser Thr Ser Asp Gly Ala Ser Ala Ala Gln Asn Ala Asp
420 425 430
Val Ala Ile Val Phe Ile Asn Ser Asp Ser Gly Glu Gly Tyr Ile Asn
435 440 445
Val Glu Gly Ser Ser Gly Asp Arg Leu Asn Leu Asp Pro Trp His Ser
450 455 460
Gly Asn Glu Leu Val Gln Ala Val Ala Gln Val Asn Gln Lys Thr Ile
465 470 475 480
Val Val Ile His Ser Val Gly Pro Leu Val Leu Glu Ser Ile Leu Ala
485 490 495
Glu Pro Asn Val Val Ala Ile Val Trp Ala Gly Leu Pro Gly Gln Glu
500 505 510
Ser Gly Asn Ala Leu Val Asp Ile Leu Tyr Gly Ser Thr Ala Pro Ser
515 520 525
Gly Lys Leu Pro Tyr Thr Ile Ala Lys Gln Glu Ser Asp Tyr Gly Thr
530 535 540
Ser Val Val Asn Gly Asp Asp Asn Phe Ser Glu Gly Ile Phe Val Asp
545 550 555 560
Tyr Arg His Phe Asp His Ala Asp Ile Glu Pro Arg Tyr Glu Phe Gly
565 570 575
Tyr Gly Leu Ser Tyr Thr Thr Phe Asn Tyr Ser Gly Leu Ala Val Asp
580 585 590
Val Thr Val Ser Ala Gly Ala Thr Ser Gly Glu Thr Val Ser Gly Gly
595 600 605
Pro Ser Asp Leu Phe Thr Glu Val Gly Thr Val Ser Ala Ser Val Gln
610 615 620
Asn Thr Gly Gln Val Thr Gly Ala Glu Val Ala Gln Leu Tyr Ile Gly
625 630 635 640
Leu Pro Ser Ser Ala Pro Ser Ala Pro Pro Lys Gln Leu Arg Gly Phe
645 650 655
Gln Lys Ile Leu Leu Glu Ala Asp Glu Ser Asp Thr Ala Ser Phe Ser
660 665 670
Leu Thr Arg Arg Asp Leu Ser Tyr Trp Asp Thr Gln Glu Gln Lys Trp
675 680 685
Val Val Pro Ser Gly Glu Phe Ser Val Tyr Val Gly Ser Ser Ser Arg
690 695 700
Asp Ile Arg Leu Thr Asp Thr Phe Thr Val
705 710
<210> 2
<211> 2145
<212> DNA
<213> Artificial sequence
<400> 2
cagacatctg attgggatga ggcatactcc aaggcactcg actccctcgc caagctcagt 60
cagaatgaaa agataggcat cgtgacggga acaagttggc agaacggttc ttgtgtaggc 120
aacacgtatc agccttcctc aatcgactac ccgtctctct gtctccaaga tggccctctt 180
ggtatccgat atgcaaaccc cgtcacggct tttcccgccg gaatcaatgc gggagctacc 240
tgggatcgct ctttgattaa agaccgcggt gctgctctag gagaagaagc aaagtctctt 300
ggggttcatg tatccttggg ccctgttgca ggaccgctgg gcaaagtgcc tcaaggagga 360
agactgtggg aaggattttc ggtcgaccca tatctcagtg gagtcgccat gacggaaacg 420
atcaacggtg ttcagggtgc tggagcccag gcgtgcgcga aacattatat tgggaatgaa 480
caggaaacga atcgcaacta catcgattcg accattgacg accgtgcctt tcatgaactc 540
tacctctggc cctttgcgga tgctgtccgc gcaaacgtcg ccagcgtgat gtgctcgtat 600
aataaagtga atggtactta tacctgtgag aatcctgcag tgctcaatca cactcttaaa 660
acagaactgg gattcaaagg ctacataatg agcgactggg gcgcgcaaca cactacaagc 720
ggcagtgcta acgctggcct ggatatgacc atgcctggaa gtgatctgag taatcctccc 780
ggtaatgttc tgtggggtca gaagcttgca gacgcgattt cgaatggaga agttgaacag 840
tctagattgg acgacatggt aacccgaatt cttgcagctt ggtatcttgt tggccaggat 900
caaggctacc catctgtaca gttcaactcg tggaatggcg gccagacctc agcgaatgtg 960
acaggagacc atgccacggt tgtccgtaat gtggcgaggg attcgattgt gctgctgaag 1020
aatgataaca atactcttcc tctctctaag ccaaacagcc tggctatcat cggaagtgat 1080
gcggcagtga accccgacgg tccaaatgcc tgtagtgatc ggggctgtga tactggaact 1140
ctggcaatgg gctggggaag tggaacatgc gagttccctt accttgttgg tccgttggaa 1200
gccattaaaa accaggcaaa tgcggatgga acaacaatca cctccagtac cacggattca 1260
actagcgatg gtgcttcagc tgctcagaac gctgacgtgg ccattgtatt tatcaattcg 1320
gattcgggtg aaggttacat caatgttgaa ggcagctctg gtgaccgcct caaccttgac 1380
ccatggcact ccggcaacga attggtccaa gcggtcgccc aggtcaacca gaagaccatc 1440
gtggtgattc acagtgttgg ccctcttgtt ctagaatcaa tcctcgcaga accgaacgtg 1500
gttgccattg tttgggctgg tctccctggt caagaaagcg gcaatgcatt ggttgatatt 1560
ctgtacggat caacagcccc aagcggaaag ttgccgtata caattgccaa gcaggagtcg 1620
gattatggca cttctgtcgt caatggcgac gataactttt ctgagggaat tttcgtcgac 1680
tatcgtcact ttgatcacgc ggatattgaa cctagatacg aatttggcta cgggttgtca 1740
tacaccacgt tcaactactc tggtctcgct gttgatgtga ctgtctcagc tggtgctacc 1800
tctggggaaa ctgtctccgg aggcccatca gatttgttca cggaagtggg taccgtctca 1860
gccagtgtac aaaacaccgg acaggtgacg ggtgctgagg tcgcccaatt gtatatcggt 1920
ttgccctcat cggcaccatc tgcaccgccg aaacagctgc gaggctttca gaaaatcctc 1980
ctcgaagccg atgagtctga cactgcgagc ttctcgctca ccagaagaga ccttagctat 2040
tgggatacac aggaacagaa atgggtggtg cccagtggcg agttctcggt atacgttgga 2100
agctccagcc gcgatattcg actaacagac actttcactg tttga 2145
<210> 3
<211> 33
<212> DNA
<213> Artificial sequence
<400> 3
acgggaacaa gttgggggaa cggttcttgt gta 33
<210> 4
<211> 33
<212> DNA
<213> Artificial sequence
<400> 4
tacacaagaa ccgttccccc aacttgttcc cgt 33
<210> 5
<211> 33
<212> DNA
<213> Artificial sequence
<400> 5
caagttggat gaacggttct tgtgtaggca aca 33
<210> 6
<211> 33
<212> DNA
<213> Artificial sequence
<400> 6
gaaccgttca tccaacttgt tcccgtcacg atg 33
<210> 7
<211> 33
<212> DNA
<213> Artificial sequence
<400> 7
tgtgctcgtg gaataaagtg aatggtactt ata 33
<210> 8
<211> 33
<212> DNA
<213> Artificial sequence
<400> 8
actttattcc acgagcacat cacgctggcg acg 33
<210> 9
<211> 714
<212> PRT
<213> Artificial sequence
<400> 9
Gln Thr Ser Asp Trp Asp Glu Ala Tyr Ser Lys Ala Leu Asp Ser Leu
1 5 10 15
Ala Lys Leu Ser Gln Asn Glu Lys Ile Gly Ile Val Thr Gly Thr Ser
20 25 30
Trp Gly Asn Gly Ser Cys Val Gly Asn Thr Tyr Gln Pro Ser Ser Ile
35 40 45
Asp Tyr Pro Ser Leu Cys Leu Gln Asp Gly Pro Leu Gly Ile Arg Tyr
50 55 60
Ala Asn Pro Val Thr Ala Phe Pro Ala Gly Ile Asn Ala Gly Ala Thr
65 70 75 80
Trp Asp Arg Ser Leu Ile Lys Asp Arg Gly Ala Ala Leu Gly Glu Glu
85 90 95
Ala Lys Ser Leu Gly Val His Val Ser Leu Gly Pro Val Ala Gly Pro
100 105 110
Leu Gly Lys Val Pro Gln Gly Gly Arg Leu Trp Glu Gly Phe Ser Val
115 120 125
Asp Pro Tyr Leu Ser Gly Val Ala Met Thr Glu Thr Ile Asn Gly Val
130 135 140
Gln Gly Ala Gly Ala Gln Ala Cys Ala Lys His Tyr Ile Gly Asn Glu
145 150 155 160
Gln Glu Thr Asn Arg Asn Tyr Ile Asp Ser Thr Ile Asp Asp Arg Ala
165 170 175
Phe His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Asn
180 185 190
Val Ala Ser Val Met Cys Ser Tyr Asn Lys Val Asn Gly Thr Tyr Thr
195 200 205
Cys Glu Asn Pro Ala Val Leu Asn His Thr Leu Lys Thr Glu Leu Gly
210 215 220
Phe Lys Gly Tyr Ile Met Ser Asp Trp Gly Ala Gln His Thr Thr Ser
225 230 235 240
Gly Ser Ala Asn Ala Gly Leu Asp Met Thr Met Pro Gly Ser Asp Leu
245 250 255
Ser Asn Pro Pro Gly Asn Val Leu Trp Gly Gln Lys Leu Ala Asp Ala
260 265 270
Ile Ser Asn Gly Glu Val Glu Gln Ser Arg Leu Asp Asp Met Val Thr
275 280 285
Arg Ile Leu Ala Ala Trp Tyr Leu Val Gly Gln Asp Gln Gly Tyr Pro
290 295 300
Ser Val Gln Phe Asn Ser Trp Asn Gly Gly Gln Thr Ser Ala Asn Val
305 310 315 320
Thr Gly Asp His Ala Thr Val Val Arg Asn Val Ala Arg Asp Ser Ile
325 330 335
Val Leu Leu Lys Asn Asp Asn Asn Thr Leu Pro Leu Ser Lys Pro Asn
340 345 350
Ser Leu Ala Ile Ile Gly Ser Asp Ala Ala Val Asn Pro Asp Gly Pro
355 360 365
Asn Ala Cys Ser Asp Arg Gly Cys Asp Thr Gly Thr Leu Ala Met Gly
370 375 380
Trp Gly Ser Gly Thr Cys Glu Phe Pro Tyr Leu Val Gly Pro Leu Glu
385 390 395 400
Ala Ile Lys Asn Gln Ala Asn Ala Asp Gly Thr Thr Ile Thr Ser Ser
405 410 415
Thr Thr Asp Ser Thr Ser Asp Gly Ala Ser Ala Ala Gln Asn Ala Asp
420 425 430
Val Ala Ile Val Phe Ile Asn Ser Asp Ser Gly Glu Gly Tyr Ile Asn
435 440 445
Val Glu Gly Ser Ser Gly Asp Arg Leu Asn Leu Asp Pro Trp His Ser
450 455 460
Gly Asn Glu Leu Val Gln Ala Val Ala Gln Val Asn Gln Lys Thr Ile
465 470 475 480
Val Val Ile His Ser Val Gly Pro Leu Val Leu Glu Ser Ile Leu Ala
485 490 495
Glu Pro Asn Val Val Ala Ile Val Trp Ala Gly Leu Pro Gly Gln Glu
500 505 510
Ser Gly Asn Ala Leu Val Asp Ile Leu Tyr Gly Ser Thr Ala Pro Ser
515 520 525
Gly Lys Leu Pro Tyr Thr Ile Ala Lys Gln Glu Ser Asp Tyr Gly Thr
530 535 540
Ser Val Val Asn Gly Asp Asp Asn Phe Ser Glu Gly Ile Phe Val Asp
545 550 555 560
Tyr Arg His Phe Asp His Ala Asp Ile Glu Pro Arg Tyr Glu Phe Gly
565 570 575
Tyr Gly Leu Ser Tyr Thr Thr Phe Asn Tyr Ser Gly Leu Ala Val Asp
580 585 590
Val Thr Val Ser Ala Gly Ala Thr Ser Gly Glu Thr Val Ser Gly Gly
595 600 605
Pro Ser Asp Leu Phe Thr Glu Val Gly Thr Val Ser Ala Ser Val Gln
610 615 620
Asn Thr Gly Gln Val Thr Gly Ala Glu Val Ala Gln Leu Tyr Ile Gly
625 630 635 640
Leu Pro Ser Ser Ala Pro Ser Ala Pro Pro Lys Gln Leu Arg Gly Phe
645 650 655
Gln Lys Ile Leu Leu Glu Ala Asp Glu Ser Asp Thr Ala Ser Phe Ser
660 665 670
Leu Thr Arg Arg Asp Leu Ser Tyr Trp Asp Thr Gln Glu Gln Lys Trp
675 680 685
Val Val Pro Ser Gly Glu Phe Ser Val Tyr Val Gly Ser Ser Ser Arg
690 695 700
Asp Ile Arg Leu Thr Asp Thr Phe Thr Val
705 710
<210> 10
<211> 714
<212> PRT
<213> Artificial sequence
<400> 10
Gln Thr Ser Asp Trp Asp Glu Ala Tyr Ser Lys Ala Leu Asp Ser Leu
1 5 10 15
Ala Lys Leu Ser Gln Asn Glu Lys Ile Gly Ile Val Thr Gly Thr Ser
20 25 30
Trp Met Asn Gly Ser Cys Val Gly Asn Thr Tyr Gln Pro Ser Ser Ile
35 40 45
Asp Tyr Pro Ser Leu Cys Leu Gln Asp Gly Pro Leu Gly Ile Arg Tyr
50 55 60
Ala Asn Pro Val Thr Ala Phe Pro Ala Gly Ile Asn Ala Gly Ala Thr
65 70 75 80
Trp Asp Arg Ser Leu Ile Lys Asp Arg Gly Ala Ala Leu Gly Glu Glu
85 90 95
Ala Lys Ser Leu Gly Val His Val Ser Leu Gly Pro Val Ala Gly Pro
100 105 110
Leu Gly Lys Val Pro Gln Gly Gly Arg Leu Trp Glu Gly Phe Ser Val
115 120 125
Asp Pro Tyr Leu Ser Gly Val Ala Met Thr Glu Thr Ile Asn Gly Val
130 135 140
Gln Gly Ala Gly Ala Gln Ala Cys Ala Lys His Tyr Ile Gly Asn Glu
145 150 155 160
Gln Glu Thr Asn Arg Asn Tyr Ile Asp Ser Thr Ile Asp Asp Arg Ala
165 170 175
Phe His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Asn
180 185 190
Val Ala Ser Val Met Cys Ser Tyr Asn Lys Val Asn Gly Thr Tyr Thr
195 200 205
Cys Glu Asn Pro Ala Val Leu Asn His Thr Leu Lys Thr Glu Leu Gly
210 215 220
Phe Lys Gly Tyr Ile Met Ser Asp Trp Gly Ala Gln His Thr Thr Ser
225 230 235 240
Gly Ser Ala Asn Ala Gly Leu Asp Met Thr Met Pro Gly Ser Asp Leu
245 250 255
Ser Asn Pro Pro Gly Asn Val Leu Trp Gly Gln Lys Leu Ala Asp Ala
260 265 270
Ile Ser Asn Gly Glu Val Glu Gln Ser Arg Leu Asp Asp Met Val Thr
275 280 285
Arg Ile Leu Ala Ala Trp Tyr Leu Val Gly Gln Asp Gln Gly Tyr Pro
290 295 300
Ser Val Gln Phe Asn Ser Trp Asn Gly Gly Gln Thr Ser Ala Asn Val
305 310 315 320
Thr Gly Asp His Ala Thr Val Val Arg Asn Val Ala Arg Asp Ser Ile
325 330 335
Val Leu Leu Lys Asn Asp Asn Asn Thr Leu Pro Leu Ser Lys Pro Asn
340 345 350
Ser Leu Ala Ile Ile Gly Ser Asp Ala Ala Val Asn Pro Asp Gly Pro
355 360 365
Asn Ala Cys Ser Asp Arg Gly Cys Asp Thr Gly Thr Leu Ala Met Gly
370 375 380
Trp Gly Ser Gly Thr Cys Glu Phe Pro Tyr Leu Val Gly Pro Leu Glu
385 390 395 400
Ala Ile Lys Asn Gln Ala Asn Ala Asp Gly Thr Thr Ile Thr Ser Ser
405 410 415
Thr Thr Asp Ser Thr Ser Asp Gly Ala Ser Ala Ala Gln Asn Ala Asp
420 425 430
Val Ala Ile Val Phe Ile Asn Ser Asp Ser Gly Glu Gly Tyr Ile Asn
435 440 445
Val Glu Gly Ser Ser Gly Asp Arg Leu Asn Leu Asp Pro Trp His Ser
450 455 460
Gly Asn Glu Leu Val Gln Ala Val Ala Gln Val Asn Gln Lys Thr Ile
465 470 475 480
Val Val Ile His Ser Val Gly Pro Leu Val Leu Glu Ser Ile Leu Ala
485 490 495
Glu Pro Asn Val Val Ala Ile Val Trp Ala Gly Leu Pro Gly Gln Glu
500 505 510
Ser Gly Asn Ala Leu Val Asp Ile Leu Tyr Gly Ser Thr Ala Pro Ser
515 520 525
Gly Lys Leu Pro Tyr Thr Ile Ala Lys Gln Glu Ser Asp Tyr Gly Thr
530 535 540
Ser Val Val Asn Gly Asp Asp Asn Phe Ser Glu Gly Ile Phe Val Asp
545 550 555 560
Tyr Arg His Phe Asp His Ala Asp Ile Glu Pro Arg Tyr Glu Phe Gly
565 570 575
Tyr Gly Leu Ser Tyr Thr Thr Phe Asn Tyr Ser Gly Leu Ala Val Asp
580 585 590
Val Thr Val Ser Ala Gly Ala Thr Ser Gly Glu Thr Val Ser Gly Gly
595 600 605
Pro Ser Asp Leu Phe Thr Glu Val Gly Thr Val Ser Ala Ser Val Gln
610 615 620
Asn Thr Gly Gln Val Thr Gly Ala Glu Val Ala Gln Leu Tyr Ile Gly
625 630 635 640
Leu Pro Ser Ser Ala Pro Ser Ala Pro Pro Lys Gln Leu Arg Gly Phe
645 650 655
Gln Lys Ile Leu Leu Glu Ala Asp Glu Ser Asp Thr Ala Ser Phe Ser
660 665 670
Leu Thr Arg Arg Asp Leu Ser Tyr Trp Asp Thr Gln Glu Gln Lys Trp
675 680 685
Val Val Pro Ser Gly Glu Phe Ser Val Tyr Val Gly Ser Ser Ser Arg
690 695 700
Asp Ile Arg Leu Thr Asp Thr Phe Thr Val
705 710
<210> 11
<211> 714
<212> PRT
<213> Artificial sequence
<400> 11
Gln Thr Ser Asp Trp Asp Glu Ala Tyr Ser Lys Ala Leu Asp Ser Leu
1 5 10 15
Ala Lys Leu Ser Gln Asn Glu Lys Ile Gly Ile Val Thr Gly Thr Ser
20 25 30
Trp Gln Asn Gly Ser Cys Val Gly Asn Thr Tyr Gln Pro Ser Ser Ile
35 40 45
Asp Tyr Pro Ser Leu Cys Leu Gln Asp Gly Pro Leu Gly Ile Arg Tyr
50 55 60
Ala Asn Pro Val Thr Ala Phe Pro Ala Gly Ile Asn Ala Gly Ala Thr
65 70 75 80
Trp Asp Arg Ser Leu Ile Lys Asp Arg Gly Ala Ala Leu Gly Glu Glu
85 90 95
Ala Lys Ser Leu Gly Val His Val Ser Leu Gly Pro Val Ala Gly Pro
100 105 110
Leu Gly Lys Val Pro Gln Gly Gly Arg Leu Trp Glu Gly Phe Ser Val
115 120 125
Asp Pro Tyr Leu Ser Gly Val Ala Met Thr Glu Thr Ile Asn Gly Val
130 135 140
Gln Gly Ala Gly Ala Gln Ala Cys Ala Lys His Tyr Ile Gly Asn Glu
145 150 155 160
Gln Glu Thr Asn Arg Asn Tyr Ile Asp Ser Thr Ile Asp Asp Arg Ala
165 170 175
Phe His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Asn
180 185 190
Val Ala Ser Val Met Cys Ser Phe Asn Lys Val Asn Gly Thr Tyr Thr
195 200 205
Cys Glu Asn Pro Ala Val Leu Asn His Thr Leu Lys Thr Glu Leu Gly
210 215 220
Phe Lys Gly Tyr Ile Met Ser Asp Trp Gly Ala Gln His Thr Thr Ser
225 230 235 240
Gly Ser Ala Asn Ala Gly Leu Asp Met Thr Met Pro Gly Ser Asp Leu
245 250 255
Ser Asn Pro Pro Gly Asn Val Leu Trp Gly Gln Lys Leu Ala Asp Ala
260 265 270
Ile Ser Asn Gly Glu Val Glu Gln Ser Arg Leu Asp Asp Met Val Thr
275 280 285
Arg Ile Leu Ala Ala Trp Tyr Leu Val Gly Gln Asp Gln Gly Tyr Pro
290 295 300
Ser Val Gln Phe Asn Ser Trp Asn Gly Gly Gln Thr Ser Ala Asn Val
305 310 315 320
Thr Gly Asp His Ala Thr Val Val Arg Asn Val Ala Arg Asp Ser Ile
325 330 335
Val Leu Leu Lys Asn Asp Asn Asn Thr Leu Pro Leu Ser Lys Pro Asn
340 345 350
Ser Leu Ala Ile Ile Gly Ser Asp Ala Ala Val Asn Pro Asp Gly Pro
355 360 365
Asn Ala Cys Ser Asp Arg Gly Cys Asp Thr Gly Thr Leu Ala Met Gly
370 375 380
Trp Gly Ser Gly Thr Cys Glu Phe Pro Tyr Leu Val Gly Pro Leu Glu
385 390 395 400
Ala Ile Lys Asn Gln Ala Asn Ala Asp Gly Thr Thr Ile Thr Ser Ser
405 410 415
Thr Thr Asp Ser Thr Ser Asp Gly Ala Ser Ala Ala Gln Asn Ala Asp
420 425 430
Val Ala Ile Val Phe Ile Asn Ser Asp Ser Gly Glu Gly Tyr Ile Asn
435 440 445
Val Glu Gly Ser Ser Gly Asp Arg Leu Asn Leu Asp Pro Trp His Ser
450 455 460
Gly Asn Glu Leu Val Gln Ala Val Ala Gln Val Asn Gln Lys Thr Ile
465 470 475 480
Val Val Ile His Ser Val Gly Pro Leu Val Leu Glu Ser Ile Leu Ala
485 490 495
Glu Pro Asn Val Val Ala Ile Val Trp Ala Gly Leu Pro Gly Gln Glu
500 505 510
Ser Gly Asn Ala Leu Val Asp Ile Leu Tyr Gly Ser Thr Ala Pro Ser
515 520 525
Gly Lys Leu Pro Tyr Thr Ile Ala Lys Gln Glu Ser Asp Tyr Gly Thr
530 535 540
Ser Val Val Asn Gly Asp Asp Asn Phe Ser Glu Gly Ile Phe Val Asp
545 550 555 560
Tyr Arg His Phe Asp His Ala Asp Ile Glu Pro Arg Tyr Glu Phe Gly
565 570 575
Tyr Gly Leu Ser Tyr Thr Thr Phe Asn Tyr Ser Gly Leu Ala Val Asp
580 585 590
Val Thr Val Ser Ala Gly Ala Thr Ser Gly Glu Thr Val Ser Gly Gly
595 600 605
Pro Ser Asp Leu Phe Thr Glu Val Gly Thr Val Ser Ala Ser Val Gln
610 615 620
Asn Thr Gly Gln Val Thr Gly Ala Glu Val Ala Gln Leu Tyr Ile Gly
625 630 635 640
Leu Pro Ser Ser Ala Pro Ser Ala Pro Pro Lys Gln Leu Arg Gly Phe
645 650 655
Gln Lys Ile Leu Leu Glu Ala Asp Glu Ser Asp Thr Ala Ser Phe Ser
660 665 670
Leu Thr Arg Arg Asp Leu Ser Tyr Trp Asp Thr Gln Glu Gln Lys Trp
675 680 685
Val Val Pro Ser Gly Glu Phe Ser Val Tyr Val Gly Ser Ser Ser Arg
690 695 700
Asp Ile Arg Leu Thr Asp Thr Phe Thr Val
705 710
<210> 12
<211> 714
<212> PRT
<213> Artificial sequence
<400> 12
Gln Thr Ser Asp Trp Asp Glu Ala Tyr Ser Lys Ala Leu Asp Ser Leu
1 5 10 15
Ala Lys Leu Ser Gln Asn Glu Lys Ile Gly Ile Val Thr Gly Thr Ser
20 25 30
Trp Gly Asn Gly Ser Cys Val Gly Asn Thr Tyr Gln Pro Ser Ser Ile
35 40 45
Asp Tyr Pro Ser Leu Cys Leu Gln Asp Gly Pro Leu Gly Ile Arg Tyr
50 55 60
Ala Asn Pro Val Thr Ala Phe Pro Ala Gly Ile Asn Ala Gly Ala Thr
65 70 75 80
Trp Asp Arg Ser Leu Ile Lys Asp Arg Gly Ala Ala Leu Gly Glu Glu
85 90 95
Ala Lys Ser Leu Gly Val His Val Ser Leu Gly Pro Val Ala Gly Pro
100 105 110
Leu Gly Lys Val Pro Gln Gly Gly Arg Leu Trp Glu Gly Phe Ser Val
115 120 125
Asp Pro Tyr Leu Ser Gly Val Ala Met Thr Glu Thr Ile Asn Gly Val
130 135 140
Gln Gly Ala Gly Ala Gln Ala Cys Ala Lys His Tyr Ile Gly Asn Glu
145 150 155 160
Gln Glu Thr Asn Arg Asn Tyr Ile Asp Ser Thr Ile Asp Asp Arg Ala
165 170 175
Phe His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Asn
180 185 190
Val Ala Ser Val Met Cys Ser Phe Asn Lys Val Asn Gly Thr Tyr Thr
195 200 205
Cys Glu Asn Pro Ala Val Leu Asn His Thr Leu Lys Thr Glu Leu Gly
210 215 220
Phe Lys Gly Tyr Ile Met Ser Asp Trp Gly Ala Gln His Thr Thr Ser
225 230 235 240
Gly Ser Ala Asn Ala Gly Leu Asp Met Thr Met Pro Gly Ser Asp Leu
245 250 255
Ser Asn Pro Pro Gly Asn Val Leu Trp Gly Gln Lys Leu Ala Asp Ala
260 265 270
Ile Ser Asn Gly Glu Val Glu Gln Ser Arg Leu Asp Asp Met Val Thr
275 280 285
Arg Ile Leu Ala Ala Trp Tyr Leu Val Gly Gln Asp Gln Gly Tyr Pro
290 295 300
Ser Val Gln Phe Asn Ser Trp Asn Gly Gly Gln Thr Ser Ala Asn Val
305 310 315 320
Thr Gly Asp His Ala Thr Val Val Arg Asn Val Ala Arg Asp Ser Ile
325 330 335
Val Leu Leu Lys Asn Asp Asn Asn Thr Leu Pro Leu Ser Lys Pro Asn
340 345 350
Ser Leu Ala Ile Ile Gly Ser Asp Ala Ala Val Asn Pro Asp Gly Pro
355 360 365
Asn Ala Cys Ser Asp Arg Gly Cys Asp Thr Gly Thr Leu Ala Met Gly
370 375 380
Trp Gly Ser Gly Thr Cys Glu Phe Pro Tyr Leu Val Gly Pro Leu Glu
385 390 395 400
Ala Ile Lys Asn Gln Ala Asn Ala Asp Gly Thr Thr Ile Thr Ser Ser
405 410 415
Thr Thr Asp Ser Thr Ser Asp Gly Ala Ser Ala Ala Gln Asn Ala Asp
420 425 430
Val Ala Ile Val Phe Ile Asn Ser Asp Ser Gly Glu Gly Tyr Ile Asn
435 440 445
Val Glu Gly Ser Ser Gly Asp Arg Leu Asn Leu Asp Pro Trp His Ser
450 455 460
Gly Asn Glu Leu Val Gln Ala Val Ala Gln Val Asn Gln Lys Thr Ile
465 470 475 480
Val Val Ile His Ser Val Gly Pro Leu Val Leu Glu Ser Ile Leu Ala
485 490 495
Glu Pro Asn Val Val Ala Ile Val Trp Ala Gly Leu Pro Gly Gln Glu
500 505 510
Ser Gly Asn Ala Leu Val Asp Ile Leu Tyr Gly Ser Thr Ala Pro Ser
515 520 525
Gly Lys Leu Pro Tyr Thr Ile Ala Lys Gln Glu Ser Asp Tyr Gly Thr
530 535 540
Ser Val Val Asn Gly Asp Asp Asn Phe Ser Glu Gly Ile Phe Val Asp
545 550 555 560
Tyr Arg His Phe Asp His Ala Asp Ile Glu Pro Arg Tyr Glu Phe Gly
565 570 575
Tyr Gly Leu Ser Tyr Thr Thr Phe Asn Tyr Ser Gly Leu Ala Val Asp
580 585 590
Val Thr Val Ser Ala Gly Ala Thr Ser Gly Glu Thr Val Ser Gly Gly
595 600 605
Pro Ser Asp Leu Phe Thr Glu Val Gly Thr Val Ser Ala Ser Val Gln
610 615 620
Asn Thr Gly Gln Val Thr Gly Ala Glu Val Ala Gln Leu Tyr Ile Gly
625 630 635 640
Leu Pro Ser Ser Ala Pro Ser Ala Pro Pro Lys Gln Leu Arg Gly Phe
645 650 655
Gln Lys Ile Leu Leu Glu Ala Asp Glu Ser Asp Thr Ala Ser Phe Ser
660 665 670
Leu Thr Arg Arg Asp Leu Ser Tyr Trp Asp Thr Gln Glu Gln Lys Trp
675 680 685
Val Val Pro Ser Gly Glu Phe Ser Val Tyr Val Gly Ser Ser Ser Arg
690 695 700
Asp Ile Arg Leu Thr Asp Thr Phe Thr Val
705 710

Claims (11)

1. A β -glucosidase mutant, wherein the β -glucosidase mutant is: mutating 34 th position of beta-glucosidase with amino acid sequence shown as SEQ ID NO.1 from glutamine to glycine; or the 34 th site of the beta-glucosidase with the amino acid sequence shown as SEQ ID NO.1 is obtained by mutating glutamine into methionine; or the 200 th site of the beta-glucosidase with the amino acid sequence shown as SEQ ID NO.1 is obtained by mutating tyrosine into phenylalanine; or the 34 th site of the beta-glucosidase with the amino acid sequence shown as SEQ ID NO.1 is mutated from glutamine into glycine, and the 200 th site of the beta-glucosidase with the amino acid sequence shown as SEQ ID NO.1 is mutated from tyrosine into phenylalanine.
2. A gene encoding the β -glucosidase mutant of claim 1.
3. An expression vector carrying the gene of claim 2.
4. A microbial cell carrying the gene of claim 2 or the expression vector of claim 3.
5. A method for preparing gentiooligosaccharide by compounding multiple enzymes is characterized in that cellulase and the beta-glucosidase mutant of claim 1 are added into a reaction system containing cellulose and glucose to react to obtain a reaction solution; and separating the reaction solution to obtain the gentiooligosaccharide.
6. The method for preparing gentiooligosaccharide by multienzyme compounding according to claim 5, wherein the cellulase is one or more of endocellulase, β -glucanase, cellobiohydrolase type I, cellobiohydrolase type II.
7. The method for preparing gentiooligosaccharide by multienzyme compounding as claimed in claim 5 or 6, wherein the concentration of cellulase added to the reaction system is 10-50U/g cellulose filter paper enzyme activity.
8. The method for preparing gentiooligosaccharides by multienzyme complex formulation as claimed in claim 5 or 6, wherein the concentration of the β -glucosidase mutant added to the reaction system is 100-400U/g glucose.
9. The method for preparing gentiooligosaccharide by multienzyme compounding according to claim 7, wherein the concentration of the substrate cellulose in the reaction system is 200 g/L.
10. The method for preparing gentiooligosaccharide by multienzyme compounding according to claim 7, wherein the concentration of substrate glucose in the reaction system is 100g/L-400 g/L.
11. Use of a mutant according to claim 1, or a gene according to claim 2, or an expression vector according to claim 3, or a microbial cell according to claim 4, or a method according to any one of claims 5 to 10 for the preparation of an oligogentiose.
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